Toxicity of titanium dioxide nanoparticles to rainbow trout

Page created by George Wolfe
 
CONTINUE READING
Toxicity of titanium dioxide nanoparticles to rainbow trout
Aquatic Toxicology 84 (2007) 415–430

                   Toxicity of titanium dioxide nanoparticles to rainbow trout
                     (Oncorhynchus mykiss): Gill injury, oxidative stress,
                                  and other physiological effects
                                  Gillian Federici, Benjamin J. Shaw, Richard D. Handy ∗
                                       Ecotoxicology and Stress Biology Research Group, School of Biological Sciences,
                                                University of Plymouth, Drake Circus, Plymouth PL4 8AA, UK
                                     Received 19 May 2007; received in revised form 10 July 2007; accepted 10 July 2007

Abstract
   Mammalian and in vitro studies have raised concerns about the toxicity of titanium dioxide nanoparticles (TiO2 NPs), but there are very limited
data on ecotoxicity to aquatic life. This paper is an observational study where we aim to describe the toxicity of TiO2 NPs to the main body systems
of rainbow trout. Stock solutions of dispersed TiO2 NPs were prepared by sonication without using solvents. A semi-static test system was used
to expose rainbow trout to either a freshwater control, 0.1, 0.5, or 1.0 mg l−1 TiO2 NPs for up to 14 days. Exposure to TiO2 NPs caused some gill
pathologies including oedema and thickening of the lamellae. No major haematological or blood disturbances were observed in terms of red and
white blood cell counts, haematocrit values, whole blood haemoglobin, and plasma Na+ or K+ concentrations. Tissue metal levels (Na+ , K+ , Ca2+
and Mn) were generally unaffected. However, some exposure concentration-dependent changes in tissue Cu and Zn levels were observed, especially
in the brain. Exposure to TiO2 NPs caused statistically significant decreases in Na+ K+ -ATPase activity (ANOVA, P < 0.05) in the gills and intestine,
and a trend of decreasing enzyme activity in the brain (the latter was not statistically significant). Thiobarbituric acid reactive substances (TBARS)
showed exposure concentration-dependent and statistically significant (ANOVA or Kruskal–Wallis test, P < 0.05) increases (two-fold or more) in
the gill, intestine and brain, but not the liver during exposure to TiO2 NPs compared to controls. TiO2 NP exposure caused statistically significant
(ANOVA, P < 0.05) increases in the total glutathione levels in the gills, but depletion of hepatic glutathione compared to controls. Total glutathione
levels in the brain and intestine were unaffected. Liver cells exposed to TiO2 NPs showed minor fatty change and lipidosis, and some hepatocytes
showed condensed nuclear bodies (apoptotic bodies). Fish probably ingested water containing TiO2 NPs during exposure (stress-induced drinking)
which may have resulted in some areas of erosion on the intestinal epithelium. Overall we conclude that titanium dioxide nanoparticles are not a
major ionoregulatory toxicant, or haemolytic, at the concentration and exposure times used. Respiratory distress is a concern and sub-lethal toxicity
involves oxidative stress, organ pathologies, and the induction of anti-oxidant defences, such as glutathione.
© 2007 Published by Elsevier B.V.

Keywords: Titanium dioxide nanoparticles; Gill; Intestine; Haematology; Na+ K+ -ATPase; TBARS; Glutathione; Rainbow trout; Copper; Zinc

1. Introduction                                                                  small size, shape, surface area, conductivity or surface chemistry
                                                                                 (Masciangioli and Zhang, 2003; Oberdörster et al., 2005) and
    Manufactured nanomaterials have been defined as new mate-                    have found numerous applications in textiles, electronics, engi-
rials with at least one dimension between 1 and 100 nm                           neering and medicine (Masciangioli and Zhang, 2003; Aitken et
(Masciangioli and Zhang, 2003). This definition is not absolute                  al., 2006). Titanium dioxide nanoparticles (TiO2 NPs) are used
and toxicological studies have included work on aggregates of                    in a range of products including sun screen, cosmetics, paint,
particles with dimensions of a few hundred nanometers (Handy                     and building materials (Aitken et al., 2006).
and Shaw, 2007a). These new materials are manufactured to have                       Most of the current literature on the toxicity on NPs comes
unique physical or chemical properties which arise from their                    from mammalian studies on respiratory exposure, or from in
                                                                                 vitro assays with mammalian cells (see Handy and Shaw, 2007a
                                                                                 for review). Studies with fine and ultrafine (
Toxicity of titanium dioxide nanoparticles to rainbow trout
416                                          G. Federici et al. / Aquatic Toxicology 84 (2007) 415–430

1985; Ferin et al., 1991; Oberdörster et al., 1992; Bermudez               Na+ K+ -ATPase activity) or oxidative stress (TBARS, gluta-
et al., 2002, 2004; Warheit et al., 2005, 2006). In particular,             thione content).
Ferin and Oberdörster (1985) demonstrated that both anastase
and rutile forms of TiO2 were toxic, and that the retention time            2. Materials and methods
was long (half times of 51–53 days in the rat lung at low mil-
ligram doses). Oberdörster et al. (1992) also showed that the              2.1. Experimental design
level of lung inflammation in rats was associated with parti-
cle size, with the smaller ultrafine TiO2 causing more adverse                  Juvenile rainbow trout (n = 189) were obtained from Hatch-
effects. Others have since confirmed these general observations             lands Trout Farm, Rattery, Devon, and held for 4 weeks in stock
in rodents. For example, Bermudez et al. (2004) exposed rats                aquaria with flowing, aerated, dechlorinated Plymouth tap water
to a 10 mg m3 aerosol of TiO2 NPs (21 nm particle size) for                 (see below). Stock animals were fed to satiation on a com-
6 weeks and found pulmonary inflammation as evidenced by                    mercial trout food. Fish weighing 28.1 ± 0.4 g (mean ± S.E.M.,
increased numbers of macrophages and neutrophils in the lung,               n = 189) were then graded into twelve experimental glass aquaria
with progressive epithelial and fibroproliferative change. Pul-             (14 fish/tank), in a triplicate design (three tanks/treatment), and
monary particle overload was also seen, with 45–57% of the                  allowed to rest for 24 h prior to the commencement of the exper-
original doses retained in the tissue 52 weeks after the exposure.          iment. Fish were exposed in triplicate to one of the following
In vitro studies also indicate some toxic effects of TiO2 NPs. Gurr         treatments for 14 days using a semi-static exposure regime (80%
et al. (2005) exposed human bronchial epithelial cells (BEAS                water change every 12 h with re-dosing after each change): con-
2B cells) to different crystal structures of TiO2 (10 or 20 nm              trol (freshwater only), 0.1, 0.5 or 1.0 mg l−1 titanium dioxide
anatase, or 200 nm rutile forms). The anatase form of TiO2 was              nanoparticles (TiO2 NPs, see below for stock solutions). These
much more toxic, inducing DNA damage, lipid peroxidation,                   concentrations of TiO2 NPs were selected after considering the
and micronuclei formation. TiO2 NPs may also be neurotoxic                  concentrations used to produce epithelial injury and oxidative
(Long et al., 2006), and can enter red blood cells in vitro (Rothen-        stress in rodents (Bermudez et al., 2004; Warheit et al., 2005,
Rutishauser et al., 2006), although the precise details of the body         2006), and the experiment was designed to allow for sub-lethal
distribution and all the target organs for TiO2 NPs in mammals              physiological effects over the exposure period rather than acute
remains uncertain.                                                          toxicity. The exposure time of 14 days was chosen to reflect
    Ecotoxicological studies with TiO2 NPs are much more                    this dosimetry and enable some physiological or biochemical
limited with a few reports on invertebrates, but almost no infor-           responses to the exposure, but also considering the ethical con-
mation on the toxic effects of TiO2 NPs to fish. Lovern and                 straint of using the minimum exposure period likely to achieve
Klaper (2006) exposed Daphnia magna to either filtered (sin-                the scientific objectives. In mammalian respiratory toxicology
gle particles of 30 nm mean diameter) or unfiltered (‘clumped’              reference materials are available to perform positive controls
particles, between 100 and 500 nm) of TiO2 NPs in a 48 h acute              with particles of known toxicity to rodent (carbon black or quartz
toxicity test. D. magna exposed to 0–10 mg l−1 filtered TiO2                particles are often used, e.g. Oberdörster et al., 1992). Reference
NPs showed increasing mortalities with increasing concentra-                particle controls for fish are not available and we therefore did
tion (100% mortality at 10 mg l−1 ); whereas 0–500 mg l−1 of the            not included these in the experimental design. The use of stan-
unfiltered TiO2 caused only 9% mortality. This study highlights             dard reference particles for fish is currently under debate along
that the type of dispersion of the TiO2 NPs may influence toxic             with other aspects of the aquatic test method (Handy and Shaw,
effect. Zhang et al. (2007) have also shown that TiO2 NPs can               2007b). It is unclear which reference materials (if any) should be
alter the uptake of other pollutants, and found that carp exposed           selected for fish, especially since historic data on reference parti-
to cadmium in the presence of TiO2 NPs accumulated 146%                     cles are not available for aquatic species, and that some materials
more Cd than controls. However, there are no detailed studies               used in mammalian studies (e.g. quartz or sand particles) may
on the toxic effects of TiO2 NPs on the different body systems              not be good positive controls given that fish species are proba-
of fish.                                                                    bly adapted to living on substrates containing large quantities of
    In the current study, we aimed to make one of the                       these materials.
first toxicological assessments of TiO2 NPs in rainbow trout                    Fish were not fed 24 h prior to, or during the experiment
(Oncorhynchus mykiss). Similar to our recent work with car-                 in order to minimise the risk of the TiO2 NPs absorbing
bon nanotubes in trout (Smith et al., 2007), we adopted a                   to food or faecal material, and to help maintain water qual-
body systems approach relating structure and function in the                ity. However, there was one exception. For ethical reasons
major organs to overview the toxic effects. Our aim was                     and to minimise aggressive behaviour associated with hunger,
to measure functional responses in key areas of physiology                  fish were fed once on day 10 just after the water change
(e.g. osmoregulation, haematology) as well as documenting                   and prior to re-dosing with TiO2 NPs. The food was eaten
organ pathologies and biochemical responses during aqueous                  immediately with no waste. Water samples were collected imme-
exposure. We therefore measured a range of end points includ-               diately before and after each water change for pH (YSI 63
ing behaviours and mortality, gill injury, haematology and                  pH meter), total ammonia (HI 95715, Hanna Instruments), and
plasma ion concentrations, trace element profiles in the major              oxygen saturation (YSI 85 D.O. meter). There were no treat-
organs, a suite of histopathological observations, as well as bio-          ment differences in water quality between tanks (ANOVA,
chemical measurements relating to physiological function (e.g.              P > 0.05). Values were (means ± S.E.M., n = 122 samples); total
Toxicity of titanium dioxide nanoparticles to rainbow trout
G. Federici et al. / Aquatic Toxicology 84 (2007) 415–430                                    417

ammonia, 0.7 ± 0.2 mg l−1 ; pH, 7.11 ± 0.01; oxygen saturation,             solutions were confirmed (Fig. 1), and renewing the test media
87.0 ± 0.25%; temperature, 14 ± 1 ◦ C. Photoperiod was 12 h                 every day would maintain the exposure, we also attempted to
light:12 h dark. The electrolyte composition of the dechlorinated           measure TiO2 NP levels in each fish tank. Water samples were
Plymouth tap water used was 0.5, 0.1, 0.4, and 0.1 mmol l−1                 analysed by measuring the absorbance peak associated with dis-
for Na+ , K+ Ca2+ and Mg2+ , respectively. Fish were randomly               persed TiO2 NPs at 329 nm (Philips PU 8720 UV/VIS Scanning
sampled on days 0 (initial fish from the stock), 7, and 14 for              Spectrophotometer, 2 nm band width) against 0–1000 !g l−1
haematology, plasma ions, tissue electrolytes, histopathology,              TiO2 NP standards. Measured concentrations of dispersed
and biochemistry (see below). The entire experiment was sub-                TiO2 NPs 10 min after dosing the tanks were 0.095 ± 0.006,
ject to ethical approval, and was independently monitored by a              0.490 ± 0.012, and 0.965 ± 0.021 mg l−1 TiO2 (mean ± S.E.M.,
fish health expert.                                                         n = 36/treatment) for the 0.1, 0.5, and 1.0 mg l−1 TiO2 treat-
                                                                            ments, respectively. This represents 95, 98, and 97% of the
2.2. Titanium dioxide nanoparticle stock solution and                       target values. Water samples were measured again just before
dosing                                                                      the 12 h water change to confirm that the TiO2 NPs had
                                                                            remained in solution. Pooled data per treatment gave mean mea-
   Powder form of ultrafine titanium dioxide nanoparticles were             sured TiO2 concentrations of 0.089 ± 0.006, 0.431 ± 0.008, and
donated by DeGussa AG, Lawrence Industries, Tamworth, UK,                   0.853 ± 0.014 mg l−1 (mean ± S.E.M., n = 126/treatment) over
and were the “Aeroxide” P25 TiO2 type (manufacturer’s infor-                the 12 h period, equating to 89, 85, and 86% of the expected
mation); crystal structure of 75 % rutile and 25% anatase TiO2 ,            concentrations, respectively.
purity was at least 99% TiO2 (maximum impurity was 1% Si),
and average particle size was 21 nm with a specific surface area            2.3. Haematology and blood plasma analysis
of 50 ± 15 m2 g−1 . This product was chosen because exactly the
same nanoparticles had been used in many of the previous mam-                   Haematology and blood plasma analysis was performed
malian studies (e.g. Bermudez et al., 2004). Chemical analysis              exactly as described in Smith et al. (2007). Briefly, two fish
of stock solutions revealed no metal impurities (data not shown),           were randomly collected from each tank (six fish/treatment, and
and the batch purity was high. Stock solutions of dispersed TiO2            n = 7 for initial fish) at days 0, 7 and 14 and carefully anaes-
NPs were prepared by sonication after considering the recom-                thetised with buffered MS222. Whole blood was collected via
mendations of the manufacturer and the findings of Matthews                 the caudal vein into heparinised syringes, and the fish weighed
(1990). Solvents were not needed. A series of trials exploring              and total length was recorded. Haematological measurements
the optimum pH, ionic strength, and sonication times required               included haematocrit (Hct), haemoglobin concentration (Hb),
to disperse the nanoparticles were conducted (data not shown).              and calculated mean erythrocyte cell volume (MEV) and mean
A stock solution of 10 g l−1 TiO2 NPs was prepared by dis-                  erythrocyte haemoglobin content (MEH) according to Handy
persing the nanoparticles in ultrapure water (Millipore, ion free           and Depledge (1999). Whole blood (20 !l) was fixed in Dacie’s
and unbuffered) with sonication for 6 h in a bath-type sonicator            fluid for red and white blood cell counts. The remaining blood
(35 kHz frequency, Fisherbrand FB 11010, Germany), and sub-                 was centrifuged (13,000 rpm for 2 min, Micro Centaur MSE),
sequently for a further 30 min sonication immediately prior to              and serum collected and stored at −20 ◦ C until subsequent anal-
dosing each day.                                                            ysis of plasma ions and osmometry as described in Smith et al.
   Dispersion was confirmed by transmission electron                        (2007).
microscopy (TEM, JEOL 1200EXII). The dispersion was very
good at the final working concentrations (0.1, 0.5 and 1.0 mg l−1           2.4. Tissue ion analysis
TiO2 NPs) and the measured particle size was close to the
manufacturers information (mean ± S.E.M., n = 100 images;                       Tissue ion analysis was performed as described in Smith et al.
24.1 ± 2.8 nm). In addition, and despite extensive sonication,              (2007) with minor modifications. Briefly, after blood sampling,
a few aggregates of nanoparticles were also observed in stock               fish were terminally anaesthetised with MS222 and dissected
solutions (mean ± S.E.M., n = 8, 160 ± 24 nm), although the                 for tissue ion analysis. Gill, liver, skinned muscle from the
majority were individual particles. Dispersion was also con-                flank, and whole brain were oven dried to a constant weight,
firmed by optical methods. Spectral scans of the sonicated TiO2             digested in concentrated nitric acid, then diluted to 16 ml with
solutions (250–700 nm, Perkin-Elmer UV/VIS Spectrometer,                    ultra pure (ion free) water. Samples were analysed for Ti, Zn,
Lambda Bio 20), gave the typical profile expected with a                    Cu, and Mn by inductively coupled plasma mass spectrometry
distinct peak at about 329 nm (Fig. 1), and was similar to                  (ICP-MS, Fisons Instruments, VG Plasma Quad PQ2 ICP-MS),
previous reports for TiO2 NPs (e.g. Lovern and Klaper, 2006).               and for Ca (Varian SpectrAA50 ), Na and K (F-AA/AE Spec-
   In order to achieve working concentrations of 0.1, 0.5, and              trometer, Double Beam GBC902) by flame atomic absorption
1.0 mg l−1 TiO2 NPs in the fish tanks, each tank was dosed with             spectroscopy. Analytical grade standards and reference materi-
0.2, 1.0 or 2.0 ml of the 10 g l−1 stock solution, respectively. The        als were used, and mass spectrometry samples also included
aeration and water flow in the tank dispersed each dose around              internal standards (1% cobalt and indium). For the Ti mea-
the tank within seconds in all experiments. Volumes of TiO2 NP              surements by ICP-MS, we also did a series of pilot analytical
stock were adjusted on re-dosing to reflect the 80% water change            chemistry studies to confirm the ICP-MS response to TiO2 NPs
every 12 h. Although the contents and composition of the stock              compared to Ti metal standards (data not shown). We found that
418                                                 G. Federici et al. / Aquatic Toxicology 84 (2007) 415–430

Fig. 1. Electron micrographs showing aggregated TiO2 NPs in a 10 mg l−1 stock solution prior to sonication (A) and dispersion to much smaller particle sizes after
sonication (B). Absorbance spectra from dispersed TiO2 NPs, (C) showing examples of 1.0 and 10.0 mg l−1 TiO2 solutions after sonicating for 2 h compared to clean
water from a fish tank, and Millipore water.

TiO2 NP standards, sonicated, and prepared using the Aeroxide                      domly collected from each tank (six fish/treatment, and n = 7
P25 particles described above gave good linear and repro-                          for initial fish) at days 0, 7 and 14 for biochemistry. Gill,
ducible calibrations using TiO2 NP concentrations ranging from                     liver, intestine, and whole brain were removed and immedi-
0 to 1000 !g l−1 (typical calibration, measured concentration in                   ately snap frozen in liquid nitrogen and stored at −80 ◦ C until
!g l−1 = 0.418x + 15.06, r2 = 0.98, where x is the expected con-                   required. Tissues (about 0.5 g or whole brain) were homogenised
centration) with a detection limit of 0.1 !g l−1 (or 2 nmol l−1 ) of               (Cat X520D with a T6 shaft, medium speed, Bennett & Co.,
TiO2 . The calibrations performed with TiO2 NP solutions had a                     Weston-super-Mare) in five volumes (2.5 ml) of ice-cold iso-
shallower slope than calibrations performed with Ti metal stan-                    tonic buffer (in mmol l−1 ; 300 sucrose, 0.1 ethylenediamine tetra
dards, and the most reliable results were with isotopes 48 Ti and                  acetic acid (EDTA), 20 (4-(2-hydroxyethyl)piperazine-1-ethane
49 Ti for TiO NPs, indicating that ICP-MS responds differently                     sulfonic acid (HEPES)), adjusted to pH 7.8 with a few drops
             2
to nanoparticles compared to pure metal solutions. Spike recov-                    of Tris (2-amino-2-hydroxylmethyl-1,3-propanediol)). Crude
ery tests for metal analysis gave good recoveries as previously                    homogenates were stored in 0.5 ml aliquots at −80 ◦ C until
reported (e.g. Hoyle et al., 2007).                                                required. Tissue homogenates were analysed at the end of the
                                                                                   experiment (day 14) for Na+ K+ -ATPase activity to determine
2.5. Biochemical analyses                                                          possible effects on osmoregulation, thiobarbituric acid reac-
                                                                                   tive substances (TBARS) and total glutathione content to assess
   Biochemical analyses were performed exactly as described                        oxidative stress. Assays were performed in triplicate exactly as
in Smith et al. (2007). Briefly, an additional two fish were ran-                  described in Smith et al. (2007) using 15, 40, and 20–40 !l
G. Federici et al. / Aquatic Toxicology 84 (2007) 415–430                                     419

of homogenate for Na+ K+ -ATPase activity, TBARS and glu-                   2.7. Statistical analysis
tathione content, respectively.
    Briefly, for Na+ K+ -ATPase activity each sample (15 !l, in                All data were analysed using StatGraphics Plus Version
triplicate) was dispensed into 400 !l of both a K+ -containing              5.1. No tank effects were observed within treatments in any
buffer and a K+ -free buffer (plus 1.0 mmol l−1 ouabain), then              experiments, so data were pooled by treatment for statistical
incubated at 37 ◦ C for 10 min. The reaction was stopped by                 analysis. After checking for kurtosis, skewedness and unequal
adding 1 ml of ice-cold trichloroacetic acid and 1 ml of colour             variance (Bartlett’s test), data were tested for treatment or time
reagent was added to each tube (9.6%, w/v FeSO4 ·6H2 O,                     effects by ANOVA followed by Fisher’s 95% least-squares dif-
1.15%, w/v ammonium heptamolybdate dissolved in 0.66 M                      ference, at 95% confidence limits. For non-parametric data the
H2 SO4 ), and colour allowed to develop for 20–30 min at                    Kruskal–Wallis test was used and differences located by notched
room temperature. Absorbances were measured at 630 nm                       box and whisker plots. The Student’s t-test was sometimes used
(Dynex MRX microplate reader) against 0–0.5 mmol l−1 phos-                  to explore differences between pairs of data sets at the end of
phate standards. The TBARS assay was performed using                        the experiment.
40 !l of homogenate (in triplicate), added to a well of a
96-well microtitre plate (in triplicate), containing 1 mol l−1 buty-        3. Results
lated hydroxytoluene (2,6-di-O-tert-butyl-4-methylphenol), and
the final volume was made up to 200 !l with 1 mmol l−1                      3.1. Aqueous exposure to titanium dioxide
phosphate buffered saline (pH 7.4). Following this, 50%                     nanoparticles
(w/v) trichloroacetic acid (TCA) and 1.3% (w/v) thiobar-
bituric acid (TBA) (dissolved in 0.3% (w/v) NaOH), were                         Aqueous exposure to TiO2 NPs did not cause mortality. Two
added, and the plate incubated at 60 ◦ C (60 min) and then                  fish died during the experimental period: one control fish was lost
cooled on ice. Absorbances were read at 530 and 630 nm                      due to fin nipping/aggression, and one fish exposed to 1.0 mg l−1
(Dynex MRX microplate reader), corrected for turbidity, and                 TiO2 at day 6 of exposure. The latter fish had signs of mucus
read against standards (0.5–25 nmol ml−1 1,1,3,3-tetraethoxy-               secretion on the gills, consistent with gill pathologies (Fig. 2).
propane).                                                                   In addition, some fish at the highest TiO2 concentration showed
    Briefly, for total glutathione content (GSH), 20–40 !l of tis-          loss of position holding in the water column for brief periods
sue homogenate, 20 !l of blank or standards (serial dilution                towards the end of the experiment (hanging vertically in the
of a 200 !mol l−1 reduced glutathione solution), was added in               water column for a few seconds–minute) which was indica-
triplicate to a microplate well containing 20 !l of 10 mmol l−1             tive of fatigue or abnormal buoyancy control. No other unusual
DTNB (5,5$ -dithiobis-(2-nitrobenzoic acid)), 260 !l of assay               behaviours were observed.
buffer (100 mmol l−1 K2 HPO4 , 5 mmol l−1 EDTA, pH 7.5),                        Histological examination of the gills at the end of the experi-
and 20 !l of 2 U ml−1 glutathione reductase (Sigma Chemicals,               ment (Fig. 2) showed normal anatomy in the freshwater controls,
Poole, UK). The reaction was commenced by the addition of                   with a normal background incidence of injuries on
420                                                 G. Federici et al. / Aquatic Toxicology 84 (2007) 415–430

Fig. 2. Gill morphology in trout after 14 days of exposure to (A) 0, (B) 0.1, (C) 0.5 and (D) 1.0 mg l−1 TiO2 NPs, using a semi-static exposure method. The gills of
control fish show normal histology, while some oedema (osmotic swelling) of some secondary lamellae are present in other treatments (white arrows), and swollen
mucocytes (black arrows). Fish exposed to TiO2 NPs showed some thickening of the primary lamellae that was absent in controls. This was more frequent at the
0.5 mg l−1 TiO2 concentration and with oedema led to some areas of fused and disrupted lamellae on some filaments (panel C). Scale bar 80 !m, sections were 8 !m
thick and stained with Mallory’s trichrome.

had evidence of hyperplasia on all gill filaments. This was not                     plasma Na+ or K+ , and values remained in the normal range for
observed in any of the control fish.                                                trout. However, there was a statistically significant increase in
   Total Ti concentrations in the tissues of the fish are shown in                  plasma Na+ in the highest TiO2 treatment at the end of the exper-
Table 1. Overall, there was no clear treatment or time-dependent                    iment compared to controls (ANOVA, P < 0.05), and a decrease
effects on Ti levels in the gill, liver or muscle, and only a transient             in K+ in the 0.5 mg l−1 TiO2 NP treatment compared to the
but statistically significant decrease (Kruskal–Wallis, P < 0.05)                   control (Table 2). Decreases in plasma Na+ were also generally
in brain Ti concentrations compared to initial fish (but no expo-                   noted compared to the initial fish (Table 2). There were no effects
sure concentration-effect).                                                         on plasma osmolarity (data not shown).

3.2. Haematology and plasma ions                                                    3.3. Tissue electrolytes and trace metals

   TiO2 NP exposure did not cause any major haematolog-                                 Fish tissues (gill, muscle, liver, whole brain) were analysed
ical disturbances and values remained in the normal range                           for major tissue electrolytes (Na+ , K+ , Ca2+ ) and trace elements
for trout (Table 2). There were no statistically significant                        (Cu, Zn, Mn). There were no statistically significant treatment-
treatment-dependent effects on haematology compared to con-                         dependent effects on tissue Na+ (data not shown, ANOVA or
trols, although there were some small but statistically significant                 Kruskal–Wallis test, P > 0.05), except for a depletion of muscle
(ANOVA, P < 0.05) decreases in red and white cell counts, Hb,                       Na+ at day 7 at the highest TiO2 NP treatment compared to the
and Hct compared to the initial stock fish. MEH showed a tran-                      control (!mol g−1 dry weight, mean ± S.E.M., n = 5–7: control,
sient elevation at day 7 compared to values on day 14 in all the                    448 ± 29; 1.0 mg l−1 TiO2 treatment, 317 ± 23), but this effect
TiO2 treatments, but these were not significantly different from                    was lost by the end of the experiment. Tissue K+ was unaf-
the within time point controls (Table 2). Mean erythrocyte cell                     fected by TiO2 exposure, except in the brain (Kruskal–Wallis
volume (MEV) showed no time or treatment-dependent effects,                         test, P = 0.029), where K+ levels in the tissue of fish exposed to
apart from a transient rise at day 7 compared to day 14 in the 0.1                  1.0 mg l−1 TiO2 NP showed a transient rise compared to controls
and 0.5 mg l−1 TiO2 treatments (Table 2). Similarly for plasma                      at day 7 (!mol g−1 dry weight, mean ± S.E.M., n = 5–7: control,
electrolytes, there were no overall treatment-dependent trends in                   225 ± 6; 1.0 mg l−1 TiO2 treatment, 303 ± 10). There were no
G. Federici et al. / Aquatic Toxicology 84 (2007) 415–430                                           421

Table 1
Tissue titanium concentrations in rainbow trout exposed to control (no TiO2 ), 0.1, 0.5, or 1.0 mg l−1 TiO2 NPs for up to 14 days
Tissue               Time (days)                Treatment

                                                Control                        0.1 mg l−1 TiO2                 0.5 mg l−1 TiO2        1.0 mg l−1 TiO2
Gill                  0                         0.28 ± 0.01 (7)
                      7                         0.37 ± 0.02 (6)                 0.35 ± 0.02 (6)                  0.32 ± 0.02 (6)      0.34 ± 0.02 (6)
                     14                         0.29 ± 0.02 (6)                 0.25 ± 0.05 (6)                  0.32 ± 0.03 (6)      0.25 ± 0.02 (6)
Liver                 0                         0.20 ± 0.01 (7)
                      7                         0.18 ± 0.01 (6)                 0.18 ± 0.01 (6)                  0.19 ± 0.01 (6)      0.19 ± 0.01 (6)
                     14                         0.26 ± 0.01 (6)#,*              0.26 ± 0.01 (6)#,*               0.27 ± 0.01 (6)#,*   0.24 ± 0.01 (6)#,*,D
Muscle                0                         0.20 ± 0.01 (7)
                      7                         0.40 ± 0.05 (6)                 0.34 ± 0.08 (6)                  0.36 ± 0.05 (6)      0.30 ± 0.03 (6)
                     14                         0.39 ± 0.06 (6)                 0.34 ± 0.05 (6)                  0.36 ± 0.06 (6)      0.33 ± 0.05 (6)
Brain                 0                         0.36 ± 0.02 (7)
                      7                         0.14 ± 0.03 (6)#                0.20 ± 0.03 (6)#                 0.29 ± 0.10 (6)#     0.17 ± 0.03 (6)#
                     14                         0.47 ± 0.09 (6)*                0.44 ± 0.08 (6)*                 0.42 ± 0.01 (6)*     0.42 ± 0.04 (6)*

Data are mean ± S.E.M. (n fish/treatment) expressed as !mol Ti metal g−1 dry weight of tissue.
 # Significantly different from initial fish (ANOVA or Kruskal–Wallis, P < 0.05).
 * Significantly different between day 7 and 14 within treatment (ANOVA or Kruskal–Wallis, P < 0.05).
 D Significantly different from the previous TiO concentration within row (ANOVA or Kruskal–Wallis, P < 0.05).
                                                 2

treatment-dependent differences in tissue Ca2+ levels, except in                     14, brain Cu and Zn levels had recovered with an overshoot,
the gill which showed a transient reduction in Ca2+ at day 7 in                      so that brain Cu/Zn concentrations in all the exposed fish were
the 0.5 mg l−1 treatment compared to controls (!mol g−1 dry                          higher than in the controls (Fig. 3). These effects on brain Zn
weight, mean ± S.E.M., n = 5–6: control, 767 ± 26; 0.5 mg l−1                        and Cu occurred without similar changes in bulk electrolytes,
TiO2 treatment, 616 ± 16). There were no statistically signifi-                      such as Na+ in the brain, and were accompanied by a small
cant treatment-dependent effects in tissue Mn levels, except in                      but statistically significant increase in brain water content in
the muscle and brain. In the muscle, Mn concentrations were                          the fish exposed to 1 mg l−1 TiO2 NP at the end of the exper-
lower in fish from the 1.0 mg l−1 TiO2 NP treatment com-                             iment compared to controls (Kruskal–Wallis test, P = 0.0008).
pared to the control at the end of the experiment (ANOVA,                            Brain water content at the end of the experiment was (% water,
P = 0.001, !mol g−1 dry weight, mean ± S.E.M., n = 6: con-                           mean ± S.E.M., n = 5–6); 73.9 ± 0.6, 75.9 ± 2.4, 77.3 ± 1.0,
trol, 0.41 ± 0.02; 1.0 mg l−1 TiO2 treatment, 0.31 ± 0.03). In the                   79.2 ± 0.4 for control, 0.1, 0.5 and 1.0 mg l−1 TiO2 treatments,
brain, only the 0.1 mg l−1 TiO2 NP treatment showed a statisti-                      respectively. There were also small changes in the water con-
cally significant increase compared to controls at the end of the                    tent of other tissues, although these did not follow any consistent
experiment (Kruskal–Wallis, P = 0.009, !mol g−1 dry weight,                          time or treatment-dependent pattern (ranges; gill, 75–79%; liver,
mean ± S.E.M., n = 4–6: control, 2.11 ± 0.04; 0.1 mg l−1 TiO2                        73–77%; muscle, 76–81%). However, there was a statistically
treatment, 3.59 ± 0.70).                                                             significant increase (Kruskal–Wallis test, P = 0.028) in muscle
    There were some interesting changes in tissue Zn and Cu                          water content at the highest TiO2 concentration compared to
levels (Fig. 3). The gills of fish exposed to 0.1 and 1.0 mg l−1                     controls at the end of the experiment (% water, mean ± S.E.M.,
TiO2 NP showed statistically significant decreases in Cu levels                      n = 5–6); 78.8 ± 0.4, 79.3 ± 4.5, 79.6 ± 0.4, 80.3 ± 0.2 for con-
compared to controls at the end of the experiment (day 14),                          trol, 0.1, 0.5 and 1.0 mg l−1 TiO2 treatments, respectively.
and all TiO2 NP treatments showed a transient depletion of
gill Zn on day 7 (statistically significant), which had recovered                    3.4. Na+ K+ -ATPase activity
by the end of the experiment (Fig. 3). This transient depletion
of zinc at day 7 also occurred in the muscle and brain, with                             Exposure to TiO2 NPs caused some decreases in Na+ K+ -
both these tissues showing an overshoot (statistically significant                   ATPase activity (Fig. 4). There was a concentration-dependent
increase above the controls, Kruskal–Wallis, P < 0.05) by the                        trend of decreasing Na+ K+ -ATPase activity in the gills and the
end of the experiment. Cu and Zn levels in the liver were not                        brain by the end of the experiment. Overall this trend was not
affected, apart from a statistically significant decrease in Zn lev-                 significantly different from the controls (ANOVA, P > 0.05),
els at the two highest TiO2 NP concentrations at day 7 (Fig. 3).                     although there was a significant difference between the 0.1
The most significant treatment-dependent changes in Cu and                           and 1.0 mg l−1 concentrations of TiO2 in the gill (Student’s
Zn levels were seen in the brain of exposed fish (Fig. 3). By                        t-test, P = 0.04). In the intestine, exposure to the lowest con-
day 7 fish from all TiO2 treatments showed about a two-fold                          centration of TiO2 caused a statistically significant decrease
decrease in brain Cu and Zn levels compared to initial fish or                       in Na+ K+ -ATPase activity compared to the control (Student’s
control fish (Kruskal–Wallis test, P < 0.0001). However, by day                      t-test, P = 0.027), but not at higher concentrations of TiO2 .
422
Table 2
Haematological parameters and plasma ion concentrations in rainbow trout exposed to control (no TiO2 ), 0.1, 0.5, or 1.0 mg l−1 TiO2 NPs for up to 14 days
Parameter                                                                Time (days)             Treatment

                                                                                                 Control                       0.1 mg l−1 TiO2               0.5 mg l−1 TiO2       1.0 mg l−1 TiO2

Haemoglobin (g dl−1 )                                                     0                        6.46 ± 0.41 (7)
                                                                          7                        5.46 ± 0.18 (6)               5.89 ± 0.48 (6)              6.14 ± 0.48 (6)       6.78 ± 0.77 (6)
                                                                         14                        5.39 ± 0.51 (6)               5.09 ± 0.61 (6)#             4.26 ± 0.21 (6)*,#    5.44 ± 0.42 (6)
Haematocrit (%)                                                           0                        29.0 ± 0.64 (7)
                                                                          7                        24.6 ± 1.78 (5)               23.4 ± 2.31 (6)#             26.0 ± 1.70 (6)       24.0 ± 1.64 (6)#

                                                                                                                                                                                                         G. Federici et al. / Aquatic Toxicology 84 (2007) 415–430
                                                                         14                        23.3 ± 1.20 (6)#              19.2 ± 2.27 (6)#             22.8 ± 1.15 (5)#      24.7 ± 1.10 (5)

Red blood cell count (cells ×106 mm3 )                                    0                        0.72 ± 0.03 (7)
                                                                          7                        0.51 ± 0.03 (6)#              0.38 ± 0.06 (6)#             0.55 ± 0.03 (6)D,#    0.51 ± 0.04 (6)#
                                                                         14                        0.57 ± 0.05 (6)#              0.58 ± 0.03 (6)*,#           0.65 ± 0.08 (6)       0.55 ± 0.06 (6)#

White blood cell count (cells ×103 mm3 )                                  0                      15.97 ± 1.94 (7)
                                                                          7                      10.28 ± 0.98 (6)#               8.95 ± 1.65 (6)#             9.12 ± 0.58 (6)#     11.10 ± 1.80 (6)#
                                                                         14                      10.30 ± 1.32 (6)#              11.73 ± 1.73 (6)#            11.47 ± 1.09 (6)#      8.64 ± 1.25 (5)#

Mean erythrocyte haemoglobin content (MEH, !g cell−1 )                    0                       9.08 ± 0.66 (7)
                                                                          7                      11.69 ± 0.90 (6)               12.41 ± 1.08 (4)#            11.07 ± 0.56 (6)      13.70 ± 1.68 (6)#
                                                                         14                       9.92 ± 1.35 (6)                9.05 ± 1.26 (6)*             7.02 ± 0.91 (6)*     10.25 ± 0.69 (6)*,D

Mean erythrocyte volume (MEV, nm3 )                                       0                      408.0 ± 17.5 (7)
                                                                          7                      512.7 ± 46.8 (6)#              592.3 ± 92.9 (6)             470.6 ± 22.5 (6)      489.9 ± 57.5 (6)
                                                                         14                      433.6 ± 55.2 (6)               337.8 ± 41.4 (6)*            327.5 ± 25.1 (6)*     508.1 ± 76.6 (6)D

Plasma Na+ (mmol l−1 )                                                    0                      142.3 ± 2.84 (7)
                                                                          7                      121.6 ± 7.99 (5)#              115.8 ± 4.11 (6)#            130.3 ± 1.31 (6)D     128.8 ± 4.68 (5)
                                                                         14                      117.6 ± 6.46 (5)#              122.2 ± 6.20 (6)#            126.8 ± 5.30 (5)#     135.7 ± 4.48 (6)+

Plasma K+ (mmol l−1 )                                                     0                         3.6 ± 0.20 (7)
                                                                          7                         3.9 ± 0.38 (5)                3.4 ± 0.17 (6)               3.1 ± 0.13 (6)+       3.3 ± 0.33 (5)
                                                                         14                         3.2 ± 0.32 (4)                3.4 ± 0.22 (6)               3.3 ± 0.10 (5)        3.7 ± 0.27 (6)

Data are mean ± S.E.M. (n fish/treatment).
 + Significant difference from control within rows (ANOVA, P < 0.05).
 * Significant difference between day 7 and day 14 within treatment (time-effect, ANOVA, P < 0.05).
 D Significantly different from the previous TiO concentration within row (exposure concentration-effect within time point, ANOVA, P < 0.05).
                                                   2
 # Significantly different from initial fish (stock fish at time zero, ANOVA, P < 0.05).
G. Federici et al. / Aquatic Toxicology 84 (2007) 415–430                                                    423

Fig. 3. Copper and zinc levels in the gill, liver, muscle and brain of trout after 7 and 14 days exposure to 0 (clear bar), 0.1 (grey bar), 0.5 (diagonal bars) or 1.0
(black bars) mg l−1 TiO2 NPs. Horizontal bars are initial (day 0) fish. Data are means ± S.E.M., !mol Cu or Zn g−1 dry weight of tissue, n = 6–7 fish, for Cu (panels
A–D), and Zn (panels E–H). Different letters within a time point indicate significant differences between treatments within each tissue (ANOVA or Kruskal–Wallis,
P < 0.05). # Significant time effect compared to initial fish (ANOVA or Kruskal–Wallis, P < 0.05). * Significant time effect within treatment compared to day 7 (ANOVA
or Kruskal–Wallis, P < 0.05).

3.5. TBARS and total glutathione                                                     centrations (Kruskal–Wallis, P < 0.05). However, TBARS in the
                                                                                     liver were unaffected by TiO2 NP exposure (ANOVA, P > 0.05).
   Fish exposed to TiO2 NPs generally showed an increase                                Total glutathione levels were measured in the gill, intes-
in TBARS compared to controls at the end of the experiment                           tine, whole brain and liver homogenates. Only the gill and
(Fig. 5). In the gills, exposure to 1.0 mg l−1 TiO2 caused the                       liver showed concentration-dependent changes in total glu-
greatest increase in TBARS (112% increase, statistically signif-                     tathione content (ANOVA, P < 0.05), while the intestine and
icant compared to the control, ANOVA P < 0.05). The intestine                        brain showed no effect (Fig. 6). The gills showed a statistically
also showed a clear concentration-dependent increase in TBARS                        significant rise in total glutathione content at the highest TiO2
(Kruskal–Wallis test, P < 0.01), and the brain showed elevations                     concentration only at the end of the experiment. However, in the
in TBARS above that of the control at all TiO2 exposure con-                         liver progressive, concentration-dependent glutathione deple-
424                                                  G. Federici et al. / Aquatic Toxicology 84 (2007) 415–430

                                                                                    sinusoid space, a few foci of lipidosis with minor fatty change
                                                                                    (Fig. 7). The occasional necrotic cell, cells with condensed
                                                                                    nuclear bodies that have the appearance of apoptotic bodies, and
                                                                                    cells showing nuclear division with condensed nuclear material
                                                                                    were noted, mainly at the highest TiO2 concentration (Fig. 7).
                                                                                       Histology of the intestine is shown in Fig. 8. Intestines from
                                                                                    control fish showed normal gross histology, but some injuries
                                                                                    were noted in fish exposed to titanium dioxide nanoparticles.
                                                                                    Two of the six fish examined that had been exposed to 0.1 mg l−1
                                                                                    TiO2 NPs showed some relatively minor histological change
                                                                                    with more diffuse definition of epithelial cells in places, and
                                                                                    fewer absorptive vacuoles. One of the fish showed occasional
                                                                                    vacuolation on the villi tips. Injuries were more progressive
                                                                                    in fish exposed to 0.5 mg l−1 TiO2 NPs. All six fish examined
                                                                                    showed occasional areas of erosion of the tips of the villi and
                                                                                    some fusion of villi (Fig. 8). One fish showed severe erosion of
                                                                                    the intestinal epithelium. Similar observations were made at the
                                                                                    highest TiO2 concentration (1.0 mg l−1 ) with all fish showing
                                                                                    occasional erosion of the villi tips, some fusion of the villi in
                                                                                    places, and with vacuolation present in the epithelium. Large
                                                                                    aggregates of nanoparticles were not seen in the gut sections
                                                                                    from any of the fish, but some of the sections with the most
                                                                                    severe injuries showed mucous residue in the lumen that also
                                                                                    had a milky colouration (presumably titanium dioxide mixed
                                                                                    with mucus secretions).
                                                                                       The brains of fish were removed whole, and the gross propor-
                                                                                    tions of the fore, mid and hind brain appeared normal. External
                                                                                    examination showed no evidence of gross inflammation (e.g.
                                                                                    no blistering of the dura or swelling of the bulk tissue) and no
                                                                                    evidence of cranial bleeding or blood vessel abnormality. Histo-
                                                                                    logical examination (not shown) confirmed that gross anatomy
                                                                                    was normal in all the brains examined from all treatments. Apart
                                                                                    from one fish in the 0.1 mg l−1 TiO2 NP treatment showing a few,
                                                                                    very localised individual necrotic cell bodies in one part of the
                                                                                    cerebrum, pathologies were absent. There was no evidence of
                                                                                    vacuolation, odema, cellular atrophy, or necrosis in brain tissue.
                                                                                    When blood vessels were evident in the sections, there was no
                                                                                    indication of swelling or bleeding. In sections where pituitary
Fig. 4. Na+ K+ -ATPase activity in crude homogenates from (A) gill, (B) intes-
                                                                                    gland was visible, the tissue appeared intact.
tine, and (C) whole brain of rainbow trout after 14 days exposure to 0 (clear
bar), 0.1 (grey bar), 0.5 (diagonal bars) or 1.0 mg l−1 (black bar) TiO2 NPs.       4. Discussion
Data are means ± S.E.M., n = 5–6 fish/treatment. Different letters indicate sig-
nificant differences between treatments within tissues (ANOVA or Student’s          4.1. Aqueous exposure to titanium dioxide nanoparticles
t-test to compare some individual data points, P < 0.05). Note the larger y-axis
scale in (panel C).
                                                                                    and gill injury

                                                                                       In the absence of suitable reference particle controls for fish,
tion was observed. The lowest concentration of TiO2 caused a                        we interpret toxic effects in terms of the presence or absence of
statistically significant rise in total glutathione (27% increase),                 TiO2 NPs (i.e. combined effect of TiO2 chemistry and nanome-
but the 0.5 and 1.0 mg l−1 TiO2 concentrations caused hepatic                       ter scale particle size), and only differentiate possible effects
total glutathione levels to decrease below that of the controls (37                 due to particle size alone where data are available from pre-
and 65 % decreases, respectively).                                                  viously published work on TiO2 powder. Only two mortalities
                                                                                    were observed in the experiment (one control fish due to fin nip-
3.6. Histological observations on the liver, intestine and                          ping, and one fish from the highest TiO2 NP concentration). We
brain                                                                               therefore argue that a 14 day exposure to TiO2 NP concentra-
                                                                                    tions between 0.1 and 1.0 mg l−1 is a sub-lethal experiment with
   The livers of fish from the freshwater control showed normal                     trout. The lethal concentration of TiO2 NP for trout is unknown,
histology, and fish exposed to TiO2 NP showed some loss of                          but our observations would suggest the lethal concentration
G. Federici et al. / Aquatic Toxicology 84 (2007) 415–430                                                    425

Fig. 5. Thiobarbituric acid reactive substances (TBARS) in crude homogenates from (A) gill, (B) intestine, (C) whole brain and (D) liver of rainbow trout after
14 days exposure to 0 (clear bar), 0.1 (grey bar), 0.5 (diagonal bars) or 1.0 mg l−1 (black bar) TiO2 NPs. Different letters indicate significant differences between
treatments within each tissue (ANOVA or Kruskal–Wallis, P < 0.05). Data are means ± S.E.M., n = 5–6 fish/treatment. Note the different y-axis scale for the liver
data (panel D).

is probably about an order of magnitude higher (10 mg l−1 or                         carbon nanotubes (SWCNTs) in trout. Smith et al. (2007) from
more) over this time scale. Similarly, dispersed TiO2 at con-                        our laboratory exposed trout to 0–0.5 mg l−1 SWCNTs in iden-
centrations around 0.5 mg l−1 cause only 9% mortality in D.                          tical conditions and found cumulative mortalities of five fish at
magna over 48 h (Lovern and Klaper, 2006). In mice, dietary                          the 0.5 mg l−1 SWCNT concentration during a 10-day exposure
doses of 5 g kg−1 of TiO2 NP also did not cause dose-dependent                       (compared to none over a longer time in this current study).
lethality over 2 weeks (Wang et al., 2007). Together these obser-                       The apparently low acute toxicity of dispersed TiO2 NPs does
vations suggest that large milligram or gram doses (per kg animal                    not mean there are no toxicological concerns, and we report a
weight) may be needed to cause acute toxicity of dispersed TiO2                      range of important sub-lethal effects in trout including organ
NPs to animals. TiO2 NPs are also less toxic than single walled                      pathologies, biochemical disturbances, and respiratory distress.

Fig. 6. Total glutathione content in crude homogenates from (A) gill, (B) intestine, (C) whole brain and (D) liver of rainbow trout after 14 days exposure to 0 (clear
bar), 0.1 (grey bar), 0.5 (diagonal bars) or 1.0 mg l−1 (black bar) TiO2 NPs. Different letters indicate significant differences between treatments within each tissue
(ANOVA, P < 0.05). Data are means ± S.E.M., n = 5–6 fish/treatment. Data are expressed as !mol of total glutathione g−1 wet weight of tissue. Note the different
y-axis scales.
426                                                   G. Federici et al. / Aquatic Toxicology 84 (2007) 415–430

Fig. 7. Liver morphology in trout after 14 days of exposure to (A) 0, (B) 0.1 (C) 0.5, and (D) 1.0 mg l−1 TiO2 NPs. The livers of control fish show normal histology
with sinusoid space (S) present. Livers from TiO2 -exposed fish showed some loss of sinusoid space and foci of liposis (black arrow). Some cells showed nuclear
fragments which appear to be apoptotic bodies (white arrows) or were in the early stages of necrosis/cellular atrophy. Note the area of necrotic cells (N) in (panel C).
Scale bar = 50 !m, sections were 8 !m thickness and stained with Mallory’s trichrome.

The gills showed injuries during exposure to TiO2 NPs (Fig. 2)                        et al., 2006), and this aspect may be worth future investigation
that were accompanied by behavioural changes. The loss of posi-                       in fish.
tion holding and swimming fatigue in the TiO2 NP exposed fish,
mucus secretion, and gill pathology are all consistent with res-                      4.2. Titanium accumulation during aqueous exposure
piratory toxicity. This is similar to studies in mammals where
epithelial injury and respiratory toxicity are reported during sim-                       Fish in this study did not accumulate TiO2 NPs in the internal
ilar milligram instillations of TiO2 NPs (Warheit et al., 2005,                       organs during exposure (Table 1). In fish nutrition, TiO2 powder
2006). Increased mucus secretion by the gills is a common                             has been used as a digestibility marker because it is not appre-
response to aqueous pollutants (Mallat, 1985), and we have                            ciably absorbed into the internal organs of fish (only about 1%
shown gill injury and mucus secretion in trout exposed to SWC-                        of ingested dose, Vandenberg and De La Noüe, 2001; Richter et
NTs (Smith et al., 2007). In the latter study, the respiratory                        al., 2003). The absence of accumulation in this study (Table 1)
distress was more severe with SWCNT than at the same con-                             suggests the situation may be similar with TiO2 NPs in trout. The
centrations of TiO2 used here. In the SWCNT experiment, gill                          tissue concentrations of Ti metal we report (0.1–0.5 !mol g−1
mucus precipitated SWCNT to help prevent direct exposure of                           dry weight, Table 1) are broadly similar to background lev-
the sensitive gill epithelium (Smith et al., 2007). Although we                       els in shellfish (0.04–1.34 !mol g−1 dry weight, Bustamante
did not see obvious large (mm) sized aggregates of TiO2 on gill                       and Miramand, 2005) and laboratory mice (1–4 !mol g−1 wet
mucus in this study, the swollen mucocytes (Fig. 2) and some                          weight depending on the organ, Wang et al., 2007). Zhang et al.
mucus secretion into the water indicate that mucous defences                          (2007) found Ti background levels in carp at the start of a TiO2
were active. This did not fully protect the gill as foci of oedema                    exposure of about 0.1 mg g−1 (about 2 !mol g−1 dry weight as
and areas of branchial hyperplasia were observed (Fig. 2). These                      TiO2 ). Zhang et al. (2007) also reported that 6 g carp exposed
gill pathologies are similar, but less severe, as those reported for                  to 10 mg l−1 TiO2 NP for 25 days accumulated a whole body
SWCNT in trout (Smith et al., 2007). However, unlike SWCNT                            concentration of about 3.39 mg g−1 as TiO2 (or 70 !mol g−1
exposure, TiO2 NP treatment also caused branchial aneurisms,                          as TiO2 ). However, in the same study, TiO2 levels in washed
suggesting either some local vascular wall injury in the branchial                    samples of muscle, gill and skin were much lower, suggest-
capillary bed or interruptions of capillary flow. TiO2 NPs are                        ing the whole carp body measurements included large amounts
known to cause microvascular dysfunction in rats (Nurkiewicz                          of surface bound TiO2 (surface adsorption rather than internal
G. Federici et al. / Aquatic Toxicology 84 (2007) 415–430                                                     427

Fig. 8. Histology of the intestine at the end of the experiment. (A) Fresh water control showing normal intestine, (B) intestine from a fish exposed to 0.1 mg l−1 TiO2
NPs showing normal gross morphology but with more diffuse definition of epithelial cells, (C) intestine from a fish exposed to 0.5 mg l−1 TiO2 NPs showing erosion
and fusion (F) of intestinal villi, (D) intestine from a fish exposed to 1.0 mg l−1 SWCNT showing fusion (F) of intestinal villi and areas of vacuolation (V). Scale
bar = 20 !m, sections were 8 !m thickness and stained with Mallory’s trichrome.

uptake into tissues). Such phenomena have been reported before                        remained within the normal range for trout. Haematologi-
in metal ecotoxicology (review, Handy and Eddy, 2004). For                            cal values in the current study (haemoglobin concentration,
example, aluminium (Al) is a well-known surface acting toxi-                          4–7 g dl−1 ; haematocrit, 19–29%, red cell counts 0.4–0.7 ×
cant that can produce indirect systemic toxic effects, but without                    106 cells mm3 , white cell counts 9–16 × 103 cells mm3 ) were
appreciable internal accumulation of the metal (Handy and Eddy,                       around the normal range expected in a juvenile rainbow trout and
1989, 1991). A similar situation may apply to TiO2 NPs in our                         are similar to other reports from our laboratory (haemoglobin
2-week aqueous exposure. Further experiments on the effects                           concentration, 4–6 g dl−1 ; haematocrit, 21–29%, red cell
of TiO2 in the gill microenvironment, effects on gill injury, and                     counts, 0.4–0.7 ×106 cells mm3 , white cell counts 6–11 ×
the systemic signalling of such injury is needed to verify this                       103 cells mm3 , Smith et al., 2007). The general trend of small
hypothesis.                                                                           decreases in cell counts and haematological parameters over
                                                                                      time compared to the initial fish is expected, and is associated
4.3. Haematological and ionoregulatory disturbances                                   with not feeding the animals during the experiment (e.g. Rios et
                                                                                      al., 2005).
   Despite some pathological effects on gill (Fig. 2) and some                            Although the gills showed a clear, concentration-dependent
inhibition of branchial Na+ K+ -ATPase activity (Fig. 4), there                       decline in branchial Na+ K+ -ATPase activity at the end of the
were no major disturbances to salt and water balance or                               experiment (Fig. 4), this did not result in depletion of plasma Na+
haematology (Table 2). Although there were some treatment-                            or K+ . This is partly explained by the intestinal Na+ K+ -ATPase
dependent effects, for example, a small increase in plasma                            activity which tended to compensate for effects at the gill by
Na+ at the highest TiO2 exposure concentration compared to                            showing normal activity when branchial Na+ K+ -ATPase activity
controls (Table 2), these effects were small and the values                           was low (Fig. 4). Thus, fish could maintain NaCl absorption
428                                           G. Federici et al. / Aquatic Toxicology 84 (2007) 415–430

from the gut lumen, even if the gill was partly compromised.                 Since these substrates are not normally present in the aquar-
Furthermore, plasma electrolytes are easily buffered by small                ium water, then this reaction is most likely to occur on contact
changes in tissue Na+ permeability (Wood, 1992), and a small                 with the tissue. For example, by using the normal trace endoge-
loss of Na+ from the muscle (observed at day 7) would help                   neous H2 O2 production from cells, or in the case of the gut any
maintain plasma Na+ concentrations. Overall, the blood data                  residue organic matter in the gut lumen. Sonication is known
suggest that TiO2 NPs are not a major ionoregulatory toxicant                to greatly increase the rates of these reactions in the presence
to trout, at least at the concentrations and exposure duration used          of TiO2 particles (Hirano et al., 2005), and there is a concern
here. The general lack of clear persistent effects of TiO2 NPs on            that sonication of the test solutions will inadvertently increase
the bulk electrolytes (Na+ , K+ and Ca2+ ) and tissue moisture               oxidative damage (we did not sonicate the control solutions that
content of tissues (with the possible exception of the muscle                had no added TiO2 NPs). However, this effect is unlikely in
moisture) also reflects this notion.                                         our experiment because the TiO2 NPs were sonicated in ultra-
    However, there were some effects on Cu and Zn homeosta-                  pure water before the TiO2 NPs had access to organic substrates
sis (Fig. 3). The gill, muscle, and brain in particular, showed              (the surface of the fish or natural water). Nonetheless, it may
decreases in both Cu and Zn at day 7 which was followed by                   be a useful precaution to sonicate the control solutions as well
a net increase in these metals compared to controls by the end               in future experiments. It is also worth considering that natural
of the experiment. This suggests that TiO2 exposure is caus-                 waters with large quantities of organic matter, or studies with
ing some initial disturbances to Cu and Zn regulation, but the               mixtures of pollutants (especially chlorinated organic chemi-
fish are able to recover. The biggest effects were seen in the               cals) could generate significant micromolar quantities of ROS
brain (Fig. 3). Similar effects were seen after aqueous expo-                within seconds of sonication (Hirano et al., 2005). These risks
sure to SWCNT in trout (Smith et al., 2007), where there were                of ROS generation during sonication need to balanced against
some exposure concentration-dependent losses of Zn and Cu                    the logic for dispersing the TiO2 NPs in every experiment.
from the gills, although not from any other organ. Clearly, Zn                   However, elevation of TBARS was also found in internal
and Cu homeostasis after TiO2 NP exposure requires further                   organs that had no direct access to the aquarium water. TBARS
investigation, especially in the brain.                                      increased in the brain (Fig. 5) without measurable increases in
                                                                             brain TiO2 levels, or TiO2 in other internal organs (Table 1),
4.4. Oxidative stress                                                        suggests some indirect mediation of oxidative stress to the inter-
                                                                             nal organs. This could simply be diffusion of the highly mobile
    TiO2 NP exposure caused an increase in TBARS in the                      hydroxy radical from sites of initial injury on the gills, and
gill, intestine and brain suggesting that the fish suffered from             with blood circulation in a small trout of less than 30 s (Handy
oxidative stress (Fig. 5). In vitro studies have also demonstrated           and Eddy, 2004) any ROS would be rapidly distributed around
that TiO2 NPs can cause lipid peroxidation (hamster embryo                   the body. A systemic hypoxia resulting in ROS also cannot be
fibroblast cells; Gurr et al., 2005) and this characteristic has             excluded given the pathology in the gills (Fig. 2, and discussion
been exploited by using TiO2 NPs as a bactericide (Maness et                 in Smith et al., 2007). Elevation of tissue Cu levels (Fig. 3) could
al., 1999). The TBARS values we report are consistent with                   also contribute to oxidative stress (Hoyle et al., 2007). Alterna-
previous measurements in trout (e.g. 1–10 nmol mg−1 protein,                 tively, a cascade of inflammatory responses could be initiated
Carriquiriborde et al., 2004), and with our recent study on SWC-             at the site of injury (the gill) that are rapidly mediated around
NTs (e.g. 2–8 nmol mg−1 protein in gill/intestine, Smith et al.,             the body by specific signalling pathways (e.g. by cytokines) as
2007). The elevation of TBARS in the gill and intestine does                 shown during respiratory TiO2 NP exposure in rodents (Driscoll,
not necessarily suggest that oxidative stress is the cause of gill           2000; Sayes et al., 2006). Clearly, nanoparticles do not have to
(Fig. 2) or intestinal pathology (Fig. 8). The elevation of TBARS            be internalised to generate systemic oxidative stress.
(Fig. 5) was not sufficient to deplete tissue glutathione in the
gill, intestine or brain (Fig. 6), suggesting that other anti-oxidant        4.5. Histology of the liver and brain
defences would be available to buffer some of the reactive oxy-
gen species (ROS) generated by TiO2 NP exposure. Interestingly                   Titanium dioxide nanoparticles have previously been shown
in the liver, a concentration-dependent depletion (utilisation) of           to cause apoptosis in mammalian cells (Rahman et al., 2002), and
total glutathione (Fig. 6) was associated with no increases in               in the present study the livers of some fish exposed to TiO2 NPs
TBARS (Fig. 5), suggesting that the liver was using up anti-                 showed condensed nuclear bodies (probably apoptotic bodies,
oxidant defences to prevent oxidative stress (no rise in TBARS).             Fig. 7). We made similar observations with SWCNT (Smith et
This is also consistent with the liver histology where only minor            al., 2007) and therefore the concern raised by Smith et al. (2007)
fatty change was observed (Fig. 7).                                          about cell cycle defects and the risk of tumour formation during
    The effects of TiO2 NPs on TBARS in the gills, and intestine             longer exposures to carbon nanotubes may also apply to TiO2
(see below), could be partly explained by direct contact of the              NPs. The fatty change observed in the liver (Fig. 7) was relatively
tissue with TiO2 NPs. We assume that most of the ROS genera-                 minor, and probably would not impact upon liver function in the
tion is from the catalytic chemical properties of TiO2 NPs, which            short term.
in the presence of light, can transfer electrons from substrates,                Histological examination of the main regions of the brain
such as hydrogen peroxide (H2 O2 ) or chlorinated organic com-               showed no overall gross pathology, although a few necrotic cells
pounds to generate the hydroxyl radical (Hirano et al., 2005).               were observed. However, the brain did show an increase in water
G. Federici et al. / Aquatic Toxicology 84 (2007) 415–430                                                     429

content during TiO2 NP exposure, elevation of TBARS (Fig. 5), a            carried out while Gillian Federici was studying for an MRes
downward trend in whole brain Na+ K+ -ATPase activity (Fig. 4),            degree in Aquatic Ecotoxicology. Technical assistance provided
and transient changes in K+ content. This suggests the brain               by Andrew Atfield, Mike Hockings, Trevor Worsey, and espe-
is showing the onset of biochemical disturbances that had not              cially Catherine Smith also on an MRes, is acknowledged. Dr.
yet manifested as organ pathology. TiO2 NPs have also been                 Andrew Fisher is thanked for help with ICP-MS.
shown to cause oxidative stress and injury to mouse microglial
cells in vitro (Long et al., 2006). We therefore cannot exclude
brain injury, behavioural, or neurological deficits during longer          References
exposures to TiO2 NPs.
                                                                           Aitken, R.J., Chaudhry, M.Q., Boxall, A.B.A., Hull, M., 2006. Manufacture and
                                                                               use of nanomaterials: current status in the UK and global trends. Occup.
4.6. Intestine and risk of exposure via the gut                                Med. (Oxford) 56, 300–306.
                                                                           Bermudez, E., Mangum, J.B., Asgharian, B., Wong, B.A., Reverdy, E.E.,
    Our previous work with SWCNT showed that nanomateri-                       Janszen, D.B., Hext, P.M., Warheit, D.B., Everitt, J.I., 2002. Long-term pul-
als can cause a stress-induced drinking response that resulted                 monary responses of three laboratory rodent species to subchronic inhalation
                                                                               of pigmentary titanium dioxide particles. Toxicol. Sci. 70, 86–97.
in SWCNT precipitation in the gut lumen and pathology of the               Bermudez, E., Mangum, J.B., Wong, B.A., Asgharian, B., Hext, P.M., Warheit,
mucosa (Smith et al., 2007). This also appears to be the case with             D.B., Everitt, J.I., 2004. Pulmonary responses of mice, rats, and hamsters
TiO2 NPs in trout. We observed an unusual milky colouration                    to subchronic inhalation of ultrafine titanium dioxide particles. Toxicol. Sci.
of the luminal fluid (presumably ingested TiO2 ) and intestinal                77, 347–357.
                                                                           Bustamante, P., Miramand, P., 2005. Subcellular and body distributions of 17
pathology (Fig. 8). The pathology included erosion of the villi,
                                                                               trace elements in the variegated scallop Chlamys varia from the French coast
fusion and vacuolation of the mucosa. Inflammation of the gas-                 of the Bay of Biscay. Sci. Total Environ. 337, 59–73.
tric mucosa has also been noted in mice during dietary TiO2 NP             Carriquiriborde, P., Handy, R.D., Davies, S.J., 2004. Physiological modulation
exposure (Wang et al., 2007). When these pathologies in the trout              of iron metabolism in rainbow trout (Oncorhynchus mykiss) fed low and
gut are considered, along with the elevation of TBARS in the                   high iron diets. J. Exp. Biol. 207, 75–86.
                                                                           Driscoll, K.E., 2000. TNF" and MIP-2: role in particle-induced inflammation
intestine (Fig. 5), it seems probable that nutritional performance
                                                                               and regulation by oxidative stress. Toxicol. Lett. 112/113, 177–184.
will be affected. These observations have broader implications,            Ferin, J., Oberdörster, G., 1985. Biological effects and toxicity assessment of
not only for the future safe use of nanomaterials in aquafeeds,                titanium dioxides: anastase and rutile. Am. Ind. Hyg. Assoc. J. 46, 69–72.
but also highlights the need for environmental risk assessments            Ferin, J., Oberdörster, G., Soderholm, S.C., Gelein, R., 1991. Pulmonary tissue
using dietary exposures.                                                       access of ultrafine particles. J. Aerosol Med. 4, 57–68.
                                                                           Gurr, J.R., Wang, A.S.S., Chen, C.H., Jan, K.Y., 2005. Ultrafine titanium dioxide
                                                                               particles in the absence of photoactivation can induce oxidative damage to
4.7. Conclusions                                                               human bronchial epithelial cells. Toxicology 213, 66–73.
                                                                           Handy, R.D., Depledge, M.H., 1999. Physiological responses: their measure-
    This study provides one of the first detailed overviews of                 ment and use as environmental biomarkers in ecotoxicology. Ecotoxicology
organ integrity and the physiological effects of TiO2 NPs in rain-             8, 329–349.
                                                                           Handy, R.D., Eddy, F.B., 1989. Surface absorption of aluminium by gill tissue
bow trout. Overall our findings are broadly similar to those we
                                                                               and body mucus of rainbow trout, Salmo gairdneri, at the onset of episodic
recently reported for SWCNT (Smith et al., 2007) in that a num-                exposure. J. Fish Biol. 34, 865–874.
ber of body systems are affected, although TiO2 may be a little            Handy, R.D., Eddy, F.B., 1991. Effects of inorganic cations on sodium adsorption
less toxic than SWCNT. Our data suggest that TiO2 NPs cause                    to the gill and body surface of rainbow trout, Oncorhynchus mykiss, in dilute
respiratory toxicity, and disturbances to the metabolism of some               solutions. Can. J. Fish. Aquat. Sci. 48, 1829–1837.
                                                                           Handy, R.D., Eddy, F.B., 2004. Transport of solutes across biological membranes
trace elements like Zn and Cu. Similar to the findings in mam-
                                                                               in eukaryotes: an environmental perspective. In: van Leeuwen, H.P., Köster,
mals, oxidative stress is also a main concern for trout during TiO2            W. (Eds.), Physicochemical Kinetics and Transport at Chemical-Biological
NP exposure. Notably, in both trout and rodents, these effects                 Interphases IUPAC Series. John Wiley, Chichester, pp. 337–356.
can occur without appreciable Ti accumulation in the internal              Handy, R.D., Shaw, B.J., 2007a. Toxic effects of nanoparticles and nanomateri-
organs in the short term. We should therefore be cautious when                 als: implications for public health, risk assessment and the public perception
                                                                               of nanotechnology. Health Risk Soc. 9, 125–144.
using body burden data for TiO2 NPs in exposures lasting only
                                                                           Handy, R.D., Shaw, B.J., 2007b. Ecotoxicity of nanomaterials to fish: chal-
a few days or a couple of weeks, since the fundamental assump-                 lenges for ecotoxicity testing. A learned discourse in Integr. Environ. Assess.
tion that internal accumulation causes toxic effect may not apply              Manag. 3 (3), 458–460.
over these shorter timescales. However, this is not a new eco-             Hoyle, I., Shaw, B.J., Handy, R.D., 2007. Dietary copper exposure in African
toxicological problem, and there is much we can apply from                     walking catfish, Clarias gariepinus: transient osmoregulatory disturbances
                                                                               and oxidative stress. Aquat. Toxicol. 83, 62–72.
our knowledge of surface acting metal toxins like Al (Handy
                                                                           Hirano, K., Nitta, H., Sawada, K., 2005. Effect of sonication on the photo-
and Eddy, 1991, 2004). For example, Zhang et al. (2007) have                   catalytic mineralization of some chlorinated organic compounds. Ultrason.
already used our adsorption theory to partly explain unpredicted               Sonochem. 12, 271–276.
interactions between Cd accumulation and TiO2 NPs in carp.                 Long, T.C., Saleh, N., Tilton, R.D., Lowry, G.V., Veronesi, B., 2006. Tita-
                                                                               nium dioxide (P25) produces reactive oxygen species in immortalized brain
                                                                               microglia (BV2): implications for nanoparticle neurotoxicity. Environ. Sci.
Acknowledgements
                                                                               Technol. 40, 4346–4352.
                                                                           Lovern, S.B., Klaper, R., 2006. Daphnia magna mortality when exposed to tita-
   This research was funded by a grant to R. Handy from the Nat-               nium dioxide and fullerene (C-60) nanoparticles. Environ. Toxicol. Chem.
ural Environment Research Council UK (NE/D007267/1) and                        25, 1132–1137.
You can also read